(405f) Multi-Parameter Optimization of Microfluidic Convective PCR for Rapid Portable Bioanalysis

The polymerase chain reaction (PCR) remains a gold-standard analytical technique for diagnosing and monitoring infectious diseases. Thermocycling approaches that employ Rayleigh-Bénard convection are of interest for portable PCR applications because their ability to continuously cycle reagents through denaturing, annealing, and extension temperature zones enables faster reaction speeds to be achieved while minimizing electrical power consumption. But the optimal design of convective thermocycling systems is not straightforward because the physical and chemical parameters that govern reaction performance (i.e., PCR tube geometry, temperature gradient, reaction biochemistry) display a complex and counterintuitive interrelationship. Here we report new studies that overcome this barrier by applying computational fluid dynamics (CFD) simulations to quantify the impact of geometry and amplicon GC content on PCR kinetics. We apply a coupled flow and reaction model using the CFD software package STAR-CCM+ to elucidate the flow field and reaction rate for an ensemble of PCR tube geometries. An adjustable temperature mapping function is incorporated to localize the denaturing, annealing, and extension processes within their respective temperature zones. Additional reaction pathways associated with reverse annealing and premature denaturing are also included to mimic processes likely to occur in the convective thermocycling format. A MATLAB-based analysis of hundreds of individual flow trajectories was also performed to validate the CFD results. These results enable the quantification of parameters such as the frequency of direct denaturing to annealing transits, the number of denaturing events, the average extension time, and the global DNA replication rate (doubling time). Surprisingly, we find that the locus of optimal design conditions can be expressed by a modified Rayleigh number that accounts for sidewall effects. We apply these insights to identify optimal PCR tube geometries capable of significantly enhanced performance, achieving results comparable to or surpassing those obtained using conventional instruments in a portable and greatly simplified format.